Selective nuclease digestion of messenger RNAs inside living cells via the short interfering RNA (siRNA)- triggered RNA interference pathway has become a mainstay in molecular biology to study gene function and holds promise for the development of new therapeutics. However, gaps in our understanding of the basic RNA interference mechanism and the ability of siRNAs to interact with intracellular RNA-binding proteins, particularly those involved in the innate immune response, limit the application of this promising new technology. Here we propose to develop new synthetic approaches to modified RNA oligonucleotides and to use the resulting molecules to carryout gene silencing with reduced undesirable off-target effects. This project has the following specific aims: 1) We will synthesize siRNAs bearing pendant alkyl groups projecting into either the major groove or the minor groove. The chemical modifications are designed to change the shape of the base while maintaining its ability to engage in Watson-Crick-like base pairing. These siRNA duplexes will be used to define the effects of nucleobase alkylation in gene silencing. The modifications are also expected to block sequence-dependent off-target effects mediated by Toll-like receptors and sequence- independent off-target effects from dsRBM proteins. 2) We hypothesize that anti-syn conformational changes in modified guanines will act as molecular switches to turn on (when Watson-Crick paired) or off (when Hoogsteen paired) steric blockades in the duplex RNA minor groove. We will investigate two alkylated guanine derivatives as switches, the common oxidized base lesion 8-oxoguanosine (8-oxoG) with added alkyl groups, and the acrolein alkylated purines. These modifications will be placed in the antisense strand using a Watson-Crick-paired sense strand for delivery;the antisense strand will be designed to target a complementary mRNA via Hoogsteen pairing to a purine at the site of modification. 3) We will evaluate the ability of modified siRNAs to engage in sequence-dependent and sequence-independent off-target effects in RNAi. Sequence-dependent effects will be assessed by analyzing modified siRNA duplexes containing immunostimulatory sequence motifs for their ability to stimulate immune responses in mice and in cultured murine immune cells including myeloid dendritic cells. Sequence-independent effects will be evaluated by determining the consequence of base modifications on the binding of cellular duplex RNA-binding proteins.